Microtubule motor proteins drive critical and diverse cellular functions, ranging from vesicle transport to mitosis. Cytoplasmic dynein and dynactin compose the minus end-directed motor complex, which along with plus end- directed kinesin motors, drives intracellular vesicle trafficking. In higher eukyarotes, active vesicle transport along the microtubule cytoskeleton is required for normal cellular function in both synthetic and degradative pathways as well as intracellular signaling. Here, we will focus on the interaction of dynein and kinesin motors in the active transport of vesicles along the axon. Axonal transport involves the coordinated activity of both dynein and kinesin motors, but the mechanisms regulating this coordination are not yet understood. This process can be studied with high temporal and spatial resolution in the cell, can be reconstituted with high fidelity in vitro, and is essential for normal neuronal function. We will use a battery of in vitro and cellular approaches to develop a unified model for dynein/dynactin and kinesin function in intracellular bidirectional vesicle transport. To develop this model, we propose the following specific aims: (1) what are the mechanisms regulating the engagement of dynein-driven retrograde transport? We hypothesize that the CAP-Gly domain of dynactin plays a critical role in the initiation of directed transport along the axon. We will test this hypothesis using cellular approaches including RNAi/rescue and expression of dominant negative and mutant constructs to determine the role of the CAP-Gly domain in vesicle transport, and to obtain insights into why mutations in this domain are so deleterious to neuronal function. Then, we will test this model by reconstituting this process in vitro using TIRF microscopy with single molecule resolution. (2) What are the mechanisms that coordinate bidirectional vesicle transport along microtubules? We hypothesize that both plus end-directed and minus end- directed motors are stably bound to vesicles transported along the axon, and that these motors are clustered into a motor platform that facilitates motor coordination. We will test this hypothesis using in vitro motility assays as well as biochemical and structural studies of isolated axonal transport vesicles, in order to determine the mechanistic consequences of interactions among vesicle-bound motors and their activators and adaptors. (3) What are the roles of scaffolding proteins in the coordination of motor function? We hypothesize that the scaffolding proteins Htt and JIPs 1 and 3 coordinate the function of oppositely oriented dynein and kinesin motors during axonal transport. We will investigate the roles of these scaffolding proteins in live cell assays as well as molecular/biochemical approaches to dissect the mechanisms underlying motor coordination in the cell. Together, these studies will build on recent observations to develop a more complete model for the initiation and regulation of bidirectional transport in the neuron. As recent work has demonstrated direct links between defects in dynein/dynactin function and neurodegenerative disease, it is essential that we develop a more mechanistically rigorous model of microtubule motor function in intracellular transport. PUBLIC HEALTH RELEVANCE: Neurons rely on active transport to maintain processes that can extend up to a meter in length. This transport is driven by the microtubule motor proteins dynein-dynactin and kinesin. Here we will study the mechanisms that regulate and coordinate microtubule motor function in the neuron. Our work will lead to a better understanding of this process in healthy cells, and will also provide insight into the mechanistic basis for multiple neurodegenerative diseases, including familial motor neuron disease, Perry Syndrome, and Huntington's disease.